Iranian Journal of Basic Medical Sciences Detection of Maga Gene in Klebsiella Spp. Isolated from Clinical Samples

which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Objective(s): Klebsiella infections are caused mainly by K. pneumoniae and K. oxytoca. In the last two decades, a new type of invasive Klebsiella pneumoniae which contains mu-coviscosity-associated gene (magA) has emerged. The aim of this study was to investigate the prevalence of magA gene and to detect antimicrobial susceptibility patterns of Kleb-siella spp. isolated from clinical samples. Materials and Methods: Klebsiella isolates were collected from patients admitted to referral hospitals of Hamadan, Iran, during a 12-month period from 2007 to 2008. The samples were analyzed by conventional microbiological methods and polymerase chain reaction (PCR). The hypermucoviscosity (HV) phenotype of Klebsiella isolates was characterized by formation of viscous strings >5 mm as a positive test. The susceptibility of isolates to routine antibiotics was assessed by agar disk diffusion method. Results: Out of 105 Klebsiella isolates, 96.2% was identified as K. pneumoniae and 3.8% as K. oxytoca by PCR. magA gene was detected in 4 (3.8%) isolates of K. pneumoniae. The isolates of K. oxytoca contained no magA gene. From 4 isolates with positive magA gene, two of them were HV+ and two were HV-phenotype. Overall, sixty-four isolates (60.95%) of K. pneumoniae showed an HV positive phenotype and all isolates of K. oxytoca were HV-phenotype. The most effective antibiotics against the isolates were tobramycin Conclusion: The results suggest that there is also magA associated serotype of the K. pneumoniae in this region. In addition, the presence of HV+ phenotype may not be associated with magA.


Introduction
Klebsiella infections are caused mainly by K. pneumoniae and K. oxytoca. They are opportunistic bacterial pathogens associated with nosocomial infections such as urinary tract infection (UTI), pneumonia and septicaemia (1,2). For the first time in 1998, a new type of invasive K. pneumoniae emerged in Taiwan, which was typically presented as a community-acquired primary liver abscess (3)(4)(5). Several reports, especially from the Asia Pacific region and the United States, have also shown that this pathogen has become the predominant cause of liver abscess (6)(7)(8). A new virulence gene which is mucoviscosityassociated gene A (magA), has recently been identified in this pathogen (9). magA is detected in a vast majority of K. pneumoniae liver abscess isolates and is associated with hypermucoviscosity (HV) and resistance to killing by human serum and phagocytosis (10).
Based on many recent reports all indicating the emer-gence of multi-drug resistance K. pneumoniae (11)(12)(13)(14). It seems that the determinantion of antimicrobial susceptibility patterns are essential for appropriate therapy (12). Antibiotic susceptibility pattern of isolates has not been previously determined in our region. Therefore, the aim of this study was to investigate the prevalence of magA gene in Klebsiella spp. isolated from clinical samples and to detect their antimicrobial susceptibility in Hamadan, Iran.

Materials and Methods
A cross-sectional study was conducted on 105 Klebsiella isolates collected from patients. The patients were admitted to referral hospitals in Hamadan, Iran, during a 12-month period from 2007 to 2008. They had no history of antibiotic therapy before sampling and informed consents were obtained from them.The samples were transferred to bacteriology laboratory, plated on MacConkey agar, eosin methylen blue agar, blood agar, incubated for 18-24 hr at 37°C and identified by conventional microbiological methods (15).

DNA extraction
A single colony was taken from each eosin methylen blue agar which had been incubated overnight and emulsified into 100 μl of phosphate buffer salt. After incubation for 10 min at 95°C, 50 μl of proteinase K (100 mg/l) and 150 μl of TE (1 mM EDTA/10 mM Tris, pH 7.5) were added to the suspension and incubated for a further 20 min at 37°C (16).
For PCR detection, the bacterial DNAs were extracted and amplified using primer pairs targeting specific sequences (Table 1). To identify K. pneumoniae isolates, 40 pair primers were designed by Oligo software using urease-D gene. These primers were tested by Blast software in the web and the best pair was chosen. For detecting K.oxytoca and magA, specific primers of phenylalanine X (PehX) and magA genes were chosen from literature (9,(17)(18)(19).
The hypermucoviscosity (HV) phenotype of Klebsiella isolates was also characterized and formation of viscous strings >5 mm in length showed a positive string test or a mucoviscose shape when a loop was passed through a colony (17).
In order to detect susceptibility of isolates to routine antibiotics, all isolates of K. pneumoniae and K. oxytoca were assessed by an agar disk diffusion method recommended by Clinical and Laboratory Standard Institute (21). Ten antibiotics including tobramycin (10 µg
MagA gene was detected in 4 isolates (3.8%) of K. pneu-  moniae, but none of the isolates of K. oxytoca contained magA gene. From the 4 isolates, three (75%) were obtained from blood samples and one (25%) from an abscess sample. Sixty-four isolates (60.95%) of K. pneumoniae showed an HV positive phenotype. From 4 isolates with positive magA gene, two of them were HV + and two were HVphenotype. The 4 isolates (100%) of K. oxytoca were HVphenotype.
The results of the susceptibility of 105 isolates of K. pneumoniae and K. oxytoca to ten routine antibiotics are shown in the Table 2.

Dicussion
The purpose of the study was to investigate the presence of magA gene in K. pneumoniae and K. oxytoca, which were isolated from clinical samples. In Fang's study, 52 out of 53 (98 %) K. pneumoniae isolated from liver abscess carried this specific virulence gene and the presence of one magA-negative isolate was thought to be due to patient's underlying disease of liver cirrhosis and hepatic failure. Thus, Fang et al. concluded that magA is an essential virulence gene for K. pneumoniae strains causing liver abscess and could be used as a diagnostic tool. Fang believed that magA gene is exclusivly limited to liver abscess and HV positive phenotype (9,10,22). With extension of global research in other countries such as North American countries, Singapore and Korea, on Klebsiella isolates, they showed magA gene isolated from other cases like acquired bacteraemia, sepsis, meningitis and endophthalmitis (5,6,8,17,23,24). In contrast to Fang's studies, these samples included HV + and HVphenotypes. Therefore, based on the results of the present as well as other studies, containing HV + phenotype is not a certain reason for the presence of magA gene since the HVphenotype may have magA gene, too (17,25). In our study, we found 4 positive magA gene isolates while two of them contained HV + and two were HVphenotype. Based on the data of this project, magA gene can specially belong to K. pneumoniae because we examined the K. oxytoca isolates for the presence of magA gene but none of them carry this gene. The survey of antibiotic susceptibility of K. pneumoniae and K. oxytoca showed different percentages of susceptibility to the tested antibiotics. In this study resistance to nalidixic acid, cefalotine and cefazolin was relatively high in contrast to other studies (13,14,26). In our study, tobramycin was mostly active against strains of Klebsiella followed by ceftazidime and ceftizoxime.

Conclusion
The K. pneumoniae magA positive strains exist in our area and are isolated from various samples. In addition, the presence of HV + phenotype is not associated with magA gene.